The long term objective of this research is to further our knowledge of the molecular mechanisms involved in the transmission of hormone and growth factor signals within cells. Particular attention will be paid to steps of the transmission pathways that involve protein phosphorylation- dephosphorylation reactions catalyzed by protein kinases and protein phosphatases, respectively. In one such pathway, the MAP kinase (MK) cascade, a series of protein kinases are involved. Listed sequentially, they include (1) a MAP kinase kinase (MAPKKK). At least three different kinases that fit in this category are known, the most studied one being the enzyme encoded by Raf-1; (2) MAP kinase (MAPKK), which is also referred to as MEK; (3) MAPK itself (MAPK); and (4) several MAPK- activated kinases, the most studied of which is p90rsk, or RSK. The completion of this cascade and the establishment of its linkage to growth factor receptors was achieved only recently; numerous laboratories, including the applicant's, participated in bringing this phase of research to fruition. Now that the components of the cascade have been defined, attention is shifting to questions related to the functions of this pathway, its regulation, and its interactions with other signaling systems. The specific aims of the present project are concerned with the characterization and mechanism of activation of several different forms of MAPKK, "cross-talk" between the MAPK cascade and other signaling systems, and the physiological role of the cascade in the regulation of growth, differentiation, and metabolism. Additionally, the functional and regulatory properties of a multifunctional protein serine/threonine kinase, casein kinase II (CKII), will be characterized. In pursuit of the goals, extensive use will be made of new methods for studying protein-protein interactions, including specific experiments directed toward interactions of MAP cascade components with the cytoskeleton. Functional studies concerned with the role of the cascade in cell growth, differentiation, and the cell cycle will make use of the overexpression of hyperactive and dominant negative mutant forms of several protein kinases of the cascade. Although the project emphasizes studies on the regulation and role of MAPKK, it will also include work on proposed roles of the more distal enzyme RSK. In particular, the role of RSK in the regulation of GSK-3 and one form of protein phosphatase-1, enzymes known to be important in the regulation of glycogen synthase, will be studied. New approaches will be used to solve the difficult problem as to how the activity of CKII is controlled within the cell and how this enzyme works in conjunction with GSK-3. These experiments should shed further light on basic aspects of growth factor functions and the regulation of cell growth and differentiation. This research is relevant to neoplastic disease and also to metabolic hormonal disorders such as diabetes mellitus.